Nuclear factor-κB Ligand Research Articles

Sympathetic nervous system contributes to orthodontic tooth movement by central neural regulation from hypothalamus.

Orthodontic tooth movement (OTM) is a specific treatment of malocclusion, whose regulation mechanism is still not clear. This study aimed to reveal the relationship between the sympathetic nervous system (SNS) and OTM through the construction of an OTM rat model through the utilization of orthodontic nickeltitanium coiled springs. The results indicated that the stimulation of SNS by dopamine significantly promote the OTM process represented by the much larger distance between the first and second molar compared with mere exertion of orthodontic force. Superior cervical ganglionectomy (SCGx) can alleviate this promotion effect, further proving the role of SNS in the process of OTM. Subsequently, the ability of orthodontic force to stimulate the center of the SNS was visualized by the tyrosin hydroxylase (TH) staining of neurons in ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC) of the hypothalamus, as well as the up-regulated expression of norepinephrine in local alveolar bone. Moreover, we also elucidated that the stimulation of SNS can promote osteoclast differentiation in periodontal ligament cells (PDLCs) and bone marrow-derived cells (BMCs) through regulation of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) system, thus promoting the OTM process. In conclusion, this study provided the first evidence for the involvement of the hypothalamus in the promotion effect of SNS on OTM. This work could provide a novel theoretical and experimental basis for further understanding of the molecular mechanism of OTM.

LncRNA TUG1 positively regulates osteoclast differentiation by targeting v-maf musculoaponeurotic fibrosarcoma oncogene homolog B

Osteoclast differentiation-mediates bone resorption is the key biological basis of orthodontic treatment while the specific mechanism of osteoclastogenesis remains unclear. This study aims to explore the underlying mechanism of the osteoclast differentiation from the perspective of long non-coding RNA (LncRNA). In the present study, the osteoclast differentiation of CD14+ peripheral blood mononuclear cells (PBMCs) was induced by recombinant human macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), and LncRNA TUG1 expression was dramatically elevated during this process. Functionally, the silence of TUG1 in CD14+ PBMCs decreased tartrate-resistant acid phosphatase (TRAP)-positive cell numbers and the protein levels of TRAP, nuclear factor of activated T cell c1 (NFATc1), and osteoclast-associated receptor (OSCAR), whereas increased V‐maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB) protein level. The subsequent experiments confirmed that TUG1 lessened the MafB protein level via accelerating its degradation. Then, the interference of MafB reversed the inhibitory effect of si-TUG1 on osteoclastogenesis, including increased the TRAP-positive cell numbers and up-regulated the protein levels of osteoclast markers. Finally, the in vivo experiments displayed that the increased TUG1 levels could promote tooth movement and bone resorption via facilitating osteoclast differentiation in the rat model of orthodontic tooth movement. In summary, TUG1 overexpressed during the process of osteoclast differentiation and positively regulated osteoclast differentiation by targeting MafB.

Bone Morphogenetic Protein-2 Promotes Osteoclasts-mediated Osteolysis via Smad1 and p65 Signaling Pathways.

An in vitro biological study. The aim of this study was to explore the role of bone morphogenetic protein-2 (BMP-2) in the regulation of osteoclast-mediated osteolysis, and the possible mechanism involving BMP-2 and nuclear factor-kappa B (NF-κB) signaling pathways. Recombinant human BMP-2 (rhBMP-2) has been approved as a therapeutic agent in spinal fusion and bone defect repair. However, its efficacy and clinical application are limited by associated complications including osteoclast-mediated bone resorption. The mechanism of BMP-2-induced osteolysis remains unknown. Bone marrow-derived macrophages (BMMs) were isolated from C57BL/6J mice and cultured with macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-κB Ligand (RANKL) to induce osteoclast differentiation. An in vitro bone resorption assay was performed by co-culturing BMMs and bone slides. The expression of BMP canonical and NF-κB signaling factors and their interaction during signal transduction were quantitated by reverse transcription polymerase chain reaction, Western blot analysis, confocal microscopy, and co-immunoprecipitation. BMP-2 enhanced osteoclast-mediated bone resorption via inducing osteoclast differentiation in a concentration-dependent manner. In addition, a high concentration of BMP-2 significant upregulated phosphorylation of BMP signaling factors p-Smad1/5/8 and NF-κB downstream factor p65, and promoted the degeneration of IκBα. In addition, BMP-2 induced osteoclast differentiation through coupling between BMP receptor II and RANK. High concentrations of BMP-2 enhanced osteoclast-mediated bone resorption by promoting RANKL-induced pre-osteoclast differentiation, probably by mediating the cross-talk between BMP canonical and NF-κB signaling pathways.Level of Evidence: N/A.

Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.

Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes–late endosomes–lysosomes in osteoclasts.

Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system.

Overexpression studies have been commonly used to yield significant advances in cell biology. In vitro osteoclast culturing involves the differentiation of bone marrow-derived monocyte macrophage precursors (BMMs) in medium supplemented with macrophage colony-stimulating factor and receptor activator of nuclear factor-kB ligand (RANKL) into mature osteoclasts. Retroviral vectors are the gold standards for efficient gene delivery into BMMs. While this strategy is effective in BMMs that are in the early stages of differentiation, it is ineffective in RANKL-treated BMMs such as mono- and multinucleated osteoclasts. This study attempted to enhance gene delivery into differentiated BMMs using liposome-mediated RNA transfection. BMMs were transfected with an EYFP overexpression plasmid or EYFP RNA by lipofection, or transduced with a retroviral vector expressing EYFP. EYFP expression was assessed by flow cytometry. We performed overexpression analyses using enhanced yellow fluorescent protein (EYFP). Although EYFP expression was observed 24 h after infection of BMMs with a recombinant retrovirus containing EYFP, expression of EYFP was observed within 3 h of transfection with EYFP RNA. Moreover, the efficiency of EYFP RNA for gene delivery into BMMs was comparable to that of retroviral transduction of EYFP. In contrast, while very few BMMs stimulated by RANKL for two days expressed EYFP after retroviral infection, more than half of the cells expressed EYFP after transfection with EYFP RNA. RNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.

The HGF/Met/NF-κB Pathway Regulates RANKL Expression in Osteoblasts and Bone Marrow Stromal Cells.

Multiple myeloma (MM)-induced bone disease occurs through hyperactivation of osteoclasts by several factors secreted by MM cells. MM cell-secreted factors induce osteoclast differentiation and activation via direct and indirect actions including enhanced expression of receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts and bone marrow stromal cells (BMSCs). Hepatocyte growth factor (HGF) is elevated in MM patients and is associated with MM-induced bone disease, although the mechanism by which HGF promotes bone disease remains unclear. In the present study, we demonstrated that HGF induces RANKL expression in osteoblasts and BMSCs, and investigated the mechanism of induction. We found that HGF and MM cell supernatants induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. In addition, HGF increased phosphorylation of Met and nuclear factor κB (NF-κB) in ST2 cells, MC3T3-E1 cells, or mouse BMSCs. Moreover, Met and NF-κB inhibitors suppressed HGF-induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. These results indicated that HGF promotes RANKL expression in osteoblasts and BMSCs via the Met/NF-κB signaling pathway, and Met and NF-κB inhibitors suppressed HGF-induced RANKL expression. Our findings suggest that Met and NF-κB inhibitors are potentially useful in mitigating MM-induced bone disease in patients expressing high levels of HGF.

MicroRNA-196a-5p in Extracellular Vesicles Secreted from Myoblasts Suppresses Osteoclast-like Cell Formation in Mouse Cells.

Muscle/bone interaction has been recently noted. Extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring microRNA (miRNA) to distant tissues. We previously reported that EVs secreted from C2C12 myoblasts (Myo-EVs) suppress osteoclast differentiation. In the present study, we identified 4 miRNAs in Myo-EVs that suppressed osteoclast-like cell formation in Raw264.7 cells using small RNA sequencing analysis. Among them, miR-196a-5p expression was higher in C2C12 cells compared to mouse osteoblasts and bone marrow cells. Transfection of miR-196a-5p mimic suppressed the mRNA levels of osteoclast-related genes and mitochondrial energy metabolism induced by receptor activator of nuclear factor-κB ligand in Raw264.7 cells. In contrast, miR-196a-5p mimic enhanced osteoblastic differentiation in ST-2 cells and MC3T3-E1 cells. In conclusion, we demonstrated that miR-196-5p suppresses osteoclast-like cell formation and mitochondrial energy metabolism in mouse cells, suggesting that it might be a crucial factor for muscle/bone interaction via EVs.

Zanthoxylum piperitum alleviates the bone loss in osteoporosis via inhibition of RANKL-induced c-fos/NFATc1/NF-κB pathway

BackgroundThe fruit of Zanthoxylum piperitum (ZP) is an herbal medicine as well as a spice agent in Asia to treat carminative, stomachic, anthelmintic and degenerative diseases. Z. piperitum was reported to have anti-oxidant, anti-inflammatory, anti-osteoarthritic and osteosarcoma proliferation-control effects. Purpose and Study designThis study was conducted to determine the anti-osteoporotic effects and mechanisms of action of ZP. MethodsFemale ICR mice underwent ovariectomies (OVX) and were orally administered ZP at 1, 10 and 100 mg/kg for 6 weeks. The femoral and tibial bones were assessed by dual‐energy X‐ray absorptiometry and histology to analyze the bone mineral density (BMD) and the number of osteoclasts. Raw 264.7 cells were stimulated by 100 ng/ml receptor activator of nuclear factor-κB ligand (RANKL) for 7 days in the presence of ZP. RANKL-induced signaling molecules were analyzed in osteoclasts. ResultsThe levels of femoral and tibial BMD were significantly increased by ZP administration. Serum biomarkers such as osteocalcin, calcium, alkaline phosphatase and bone-specific alkaline phosphatase concentrations were markedly recovered to normal levels in ZP-treated osteoporotic mice. In addition, the number of osteoclasts in the head, trochanter and body of the femur was obviously decreased in the ZP treatment groups. Moreover, ZP treated-cells showed a reduction in the number of TRAP-positive multinuclear cells in RANKL-stimulated Raw 264.7 cells. ZP decreased the RANKL-activated NFATc1 and c-fos, transcription factors of osteoclast formation. The nuclear translocation of NF-κB and phosphorylation of ERK42/44 were inhibited by the ZP treatment in RANKL-induced osteoclasts. ConclusionCollectively, ZP exerts its inhibitory effect against bone resorption by regulating RANKL-mediated c-fos/NFATc1/NF-κB in osteoclast. ZP may prove to be a therapeutic agent for osteoporosis.

Delineating role of NF‐κB and interacting cytokines during prostate cancer‐induced osteoclastogenesis

Prostate cancer (PCa) frequently metastasizes to the bone leading to devastating complications such as severe pain and fracture. However, the mechanisms by which PCa cells cause bone loss remain less understood. We investigated the role and mechanisms by which PCa cells induce osteoclastogenesis using cultured monocytic osteoclast precursors. Treatment of RAW264.7 cells with PCa cell lines: DU145, LNCaP, PC-3, or their conditioned media led to the formation of distinct multinucleated, TRAP+ osteoclasts. This phenomenon was associated with the increased activation of transcription factor nuclear factor-kB (NF-κB). High transcript level of receptor activator of nuclear factor-kB ligand (RANKL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were detected in PCa cells. TNF-α and LT-α augmented, whereas IL-6 reduced the RANKL-induced osteoclast formation in RAW264.7 cultures. Our results also demonstrated that PCa cells-induced osteoclastogenesis involved the activation of the TRAF6-IKK-p65-NF-κB signaling cascade. Together, our study demonstrates that PCa cells produce RANKL and several other pro-inflammatory cytokines known to influence osteoclastogenesis, by targeting the NF-κB signaling pathway.

TANK-binding kinase 1 mediates osteoclast differentiation by regulating NF-κB, MAPK and Akt signaling pathways.

TANK-binding kinase 1 (TBK1) belongs to the noncanonical IκB kinase (IKK) family. The ubiquitously expressed protein is well known to play a pivotal role in innate immune response and inflammation. Although excessive inflammatory activities have been shown to affect osteoclast (OC) differentiation and function, direct relevance of TBK1 in bone turnover is not known. In this work, we specifically altered the TBK1 protein level by knocking down or overexpressing it without affecting its homologous protein IKKε expression, and demonstrated the effect of TBK1 on OC differentiation in bone marrow macrophages (BMMs) and RAW264.7 cells upon induction by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). TBK1 knockdown was found to markedly inhibit the OC differentiation and function, while TBK1 overexpression enhanced OC formation. Downregulation of TBK1 greatly suppressed RANKL-induced gene expression of Mmp9, Atp6v0d2, Acp5, Ctsk andNfatc1 involved in the regulation of OC formation and function in both BMM and RAW264.7 cells. Mechanistic studies indicated that TBK1 affected the NF-κB signaling pathway as well as mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) activation during OC differentiation. Moreover, the protein level of TNF receptor-associated factor 6 (TRAF6) was increased, and the interaction of TRAF6 with TBK1 was potentiated, by RANKL. Collectively, we provide direct evidence showing that TBK1 effectively mediates OC differentiation and function by regulating NF-κB, MAPKs and Akt signals. A TBK1-targeted therapeutic strategy may be useful for the treatment of bone-related disorders.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

SH3P2 suppresses osteoclast differentiation through restricting membrane localization of myosin 1E.

Osteoclasts are multinucleated cells responsible for bone resorption. Src homology 3 (SH3) domain-containing protein-2 (SH3P2)/osteoclast-stimulating factor-1 regulates osteoclast differentiation, but its exact role remains elusive. Here, we show that SH3P2 suppresses osteoclast differentiation. SH3P2 knockout (KO) mice displayed decreased femoral trabecular bone mass and enhanced localization of osteoclasts on the tibial trabecular bone surface, suggesting that SH3P2 suppresses bone resorption by osteoclasts. Osteoclast differentiation based on cellular multinuclearity induced by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) was enhanced in bone marrow-derived macrophages lacking SH3P2. RANKL induced SH3P2 dephosphorylation, which increased the association of actin-dependent motor protein myosin 1E (Myo1E) with SH3P2 and thereby prevented Myo1E localization to the plasma membrane. Consistent with this, Myo1E in the membrane fraction increased in SH3P2-KO cells. Together with the attenuated osteoclast differentiation in Myo1E knocked down cells, SH3P2 may suppress osteoclast differentiation by preventing their cell-to-cell fusion depending on Myo1E membrane localization.

Oral toxicity to high level sodium fluoride causes impairment of autophagy.

Autophagy is a highly conserved intracellular digestion process that degrades damaged proteins and organelles but the biological roles of autophagy in pathological aspects of oral tissues remain largely unknown. We sought to elucidate the function of autophagy, especially its interplay with apoptosis and oxidative stress, in the oral toxicity induced by exposure to 5 mM sodium fluoride (NaF). Human cementoblasts (HCEM-2) in culture were exposed to 5 mM NaF for 5 min, after which cell viability and cell apoptosis were assessed using the MTS assay and an Annexin V-FITC/PI apoptosis detection kit, respectively. Quantitative RT-PCR and Western blotting were performed to characterize the expression levels of markers for autophagy, apoptosis, and oxidative stress. Senescence-resistant (SAMR1) mice were exposed to 5 mM NaF in their drinking water from 12 to 58 weeks. Micro-computed tomography was used to measure changes in their alveolar bone while immunohistochemistry and immunofluorescence staining was used to evaluate protein expression levels. HCEM-2 cells exposed to 5 mM NaF had decreased levels of autophagy, as shown by reduced expression levels of ATG5, Beclin-1 and LC3-II, elicited apoptosis, which in turn induced oxidative stress and inflammation, as manifested by elevated levels of Bax, cleaved caspase-3, SOD1 and phospho NF-κB. Treatment of mice with 5 mM NaF resulted in histological abnormalities in periodontal tissues, induced excessive oxidative stress and apoptosis, and reduced autophagy. Micro-computed tomography analysis demonstrated that 5 mM NaF caused a decrease in bone areas of mice compared with controls. Exposure to 5 mM NaF induced RANKL (receptor activator of nuclear factor κB ligand) and cathepsin K expression in periodontal tissues, while ATG5 and Beclin-1 expression was abrogated by 5 mM NaF. Taken together, our findings suggest that 5 mM NaF elicits oral toxicity that contributes to excessive apoptosis, oxidative stress, and defective autophagy, which aggravates periodontal tissue damage.

Osteoclast Differentiation Is Suppressed by Increased O-GlcNAcylation Due to Thiamet G Treatment.

Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.

RNA Synthesis by DNA Methyltransferase 1 siRNA Transfection with Cationic Liposomes into Osteoblastic Progenitor Cells Based on SiO² Nanomagnetic Beads for Proliferation and Differentiation.

DNA methylation regulated gene expression is important for osteoblast proliferation and differentiation during bone remodeling and its deregulation leads to the development of osteoporosis. DNA methyltransferase 1 (DNMT1) is an important regulator of DNA methylation. To explore the effect and mechanism of differential expression of DNMT1 in osteoblast precursor cells, DNMT1 siRNAs were designed and synthesized to interfere with DNMT1 expression in the osteoblast precursor cells, MC3T3E1 (Clone 24; MC3T3E1-24). The expression of the target gene, DNMT1, and osteogenic differentiation indicators osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). MTT assay was used to detect the effect on cell proliferation. Alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the effect of DNMT1 on osteogenic differentiation. Hematoxylin and eosin (H&E) staining was used to detect the morphological changes in MC3T3E1-24 cells. Twenty-four hours following the transfection of MC3T3E1-24 cells with DNMT1 siRNA using cationic liposomes, DNMT1 mRNA and protein levels decreased significantly (P <0.001 for both). The reduced expression of DNMT1 promoted the OPG mRNA and protein expression (P <0.05), increased the ratio of OPG to RANKL (P <0.05), inhibited the expression of RANKL (P <0.01) without affecting the RANKL gene expression (not significant, P >0.05). The reduced expression of DNMT1 also promoted the proliferation of osteoblast precursor cells. In addition, ALP activity test and alizarin red staining showed that reduced expression of DNMT1 resulted in an increase in OPG/RANKL ratio and promoted the differentiation of the precursor cells. The cultured cells were found to have fibroblast-like appearance, and calcium nodules were observed after 7 days of conventional culture. In addition, to improve the efficiency of RNA extraction and save time, a type of silica nanomagnetic beads was used in the early stage of this study to extract RNA and assist qPCR detection of the target genes. The results showed that the magnetic beads could effectively extract RNA from the cells. In conclusion, low expression of DNMT1 affects proliferation and maturation of osteoblasts by upregulating OPG and OPG/RANKL ratio.

Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro.

Iron overload is closely associated with osteoporosis, the potential cellular mechanism involved in decreased osteoblast differentiation and increased osteoclast formation. However, the effect of iron overload on the biological behavior in osteocytes has not been reported. This study aims to investigate the changes of osteocytic activity, apoptosis, and its regulation on osteoclastogenesis in response to iron overload. MLO-Y4 osteocyte-like cells and primary osteocytes from mice were processed with ferric ammonium citrate (FAC) and deferoxamine (DFO), the conditioned medium (CM) was harvested and co-cultured with Raw264.7 cells and bone marrow-derived macrophages (BMDMs) to induce them to differentiate into osteoclasts. Osteocyte apoptosis, osteoclast differentiation, osteocytic gene expression and protein secretion of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) was examined. Excessive iron has a toxic effect on MLO-Y4 osteocyte-like cells. Increased cell apoptosis in MLO-Y4 cells and primary osteocytes was induced by iron overload. The osteoclastic formation, differentiation-related gene expression, and osteoclastic bone-resorption capability were significantly increased after treated with the CM from iron overload-exposed osteocytes. Excessive iron exposure significantly promoted the gene expression and protein secretion of the RANKL in MLO-Y4 cells. Addition of RANKL-blocking antibody completely abolished the increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron. Moreover, the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone) was used to inhibit osteocyte apoptosis. The results showed osteocyte apoptosis induced by iron overload was reduced by QVD and accompanied by the decrease of soluble RANKL (sRANKL) in supernatant. The increase of osteoclast formation and bone resorption capacity induced by the CM from osteocytes exposed to excessive iron was significantly decreased by QVD. These results indicated that iron overload-induced osteocyte apoptosis is required to regulate osteoclast differentiation by increasing osteocytic RANKL production. This study, for the first time, reveals the indirect effect of iron overload on osteoclast differentiation through regulating osteocytes.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcPstaining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

Discovery of Small-Molecule Inhibitors of Receptor Activator of Nuclear Factor-κB Ligand with a Superior Therapeutic Index

Receptor activator of nuclear factor-κB ligand (RANKL) constitutes the master mediator of osteoclastogenesis, while its pharmaceutical inhibition by a monoclonal antibody has been approved for the treatment of postmenopausal osteoporosis. To date, the pursuit of pharmacologically more favorable approaches using low-molecular-weight inhibitors has been hampered by low specificity and high toxicity issues. This study aimed to discover small-molecule inhibitors targeting RANKL trimer formation. Through a systematic screening of 39 analogues of SPD-304, a dual inhibitor of tumor necrosis factor (TNF) and RANKL trimerization, we identified four compounds (1b, 3b, 4a, and 4c) that selectively inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, without affecting TNF activity or osteoblast differentiation. Based on structure-activity observations extracted from the most potent and less toxic inhibitors of RANKL-induced osteoclastogenesis, we synthesized a focused set of compounds that revealed three potent inhibitors (19a, 19b, and 20a) with remarkably low cell-toxicity and improved therapeutic indexes as shown by the LC50 to IC50 ratio. These RANKL-selective inhibitors are an excellent starting point for the development of small-molecule therapeutics against osteolytic diseases.

NRF2 Is an Upstream Regulator of MYC-Mediated Osteoclastogenesis and Pathological Bone Erosion.

Osteoclasts are the sole bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathogenic bone destruction such as inflammatory arthritis. Pharmacologically targeting osteoclasts has been a promising approach to alleviating bone disease, but there remains room for improvement in mitigating drug side effects and enhancing cell specificity. Recently, we demonstrated the crucial role of MYC and its downstream effectors in driving osteoclast differentiation. Despite these advances, upstream regulators of MYC have not been well defined. In this study, we identify nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor known to regulate the expression of phase II antioxidant enzymes, as a novel upstream regulator of MYC. NRF2 negatively regulates receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis through the ERK and p38 signaling-mediated suppression of MYC transcription. Furthermore, the ablation of MYC in osteoclasts reverses the enhanced osteoclast differentiation and activity in NRF2 deficiency in vivo and in vitro in addition to protecting NRF2-deficient mice from pathological bone loss in a murine model of inflammatory arthritis. Our findings indicate that this novel NRF2-MYC axis could be instrumental for the fine-tuning of osteoclast formation and provides additional ways in which osteoclasts could be therapeutically targeted to prevent pathological bone erosion.

New insight into unexpected bone formation by denosumab

Denosumab (Dmab) was the first monoclonal antibody (mAb) approved for the treatment of osteoporosis. It blocks the receptor activator for nuclear factor κB ligand (RANKL) and acts as a potent antiresorptive agent. In contrast to classic antiresorptive agents, Dmab treatment leads to a progressive increase in bone mass, but the mechanisms remain controversial. Recently, RANKL signaling in osteoblastogenesis and bone formation and RANKL reverse signaling in coupling bone resorption and formation were demonstrated. Thus, here we discuss the roles of RANKL signaling and RANKL reverse signaling in the bone-forming effects of Dmab.